Project Summary Erythropoiesis is the process by which red blood cells (RBC) are produced from erythroid progenitors. Under normal conditions more than 100 billion RBC are produced each day. However, this number must increase in response to anemic conditions caused by hypoxia, hemorrhage, hemolysis, or other conditions that reduce RBC number or hemoglobin levels in the blood. The World Health Organization estimates more than 1.6 billion people suffer from anemia worldwide leading to significant morbidity and mortality. Mechanisms regulating erythropoiesis in response to anemia, or stress erythropoiesis, are not fully understood. Signaling by steroid hormone receptors, specifically the glucocorticoid receptor, are necessary for an erythropoietic response to stress. Previous work by the Baron laboratory has identified the Vitamin D3 receptor (VDR) transcription factor as a regulator of erythroid progenitor proliferation in vitro. VDR is a member of the steroid hormone family of transcription factors. Binding of the Vitamin D3 ligand activates VDR and induces its translocation into the nucleus, where it recruits transcriptional coregulatory complexes. The VDR signaling pathway has been studied mostly in bone and in cancers. The regulation of erythropoiesis by this pathway has been essentially unexplored. The aim of this proposal is to elucidate the mechanism by which VDR signaling regulates erythropoiesis. My preliminary data suggest that VDR signaling regulates cell cylce control genes. I will first test whether VDR signaling maintains progenitors and delays their differentiation by regulating the cell cycle. I will determine whether activation of VDR in fetal liver erythroid progenitors influences cell cycle length. Using small molecule inhibitors specific for Cdk4/6, I will ask whether blocking their function in cultured fetal liver erythroid progenitors mimics the effects of VDR activation. I will determine if VDR signaling alters level of expression and/or the phosphorylation status of the cyclin D-Cdk4/6 complex target protein Rb. Next I will test the hypothesis that VDR signaling plays a role in stress erythropoiesis in vivo using mouse models. I will use a Vdr knockout mouse to test the role of VDR in response to stress. Vdr null mutant, heterozygous, and wild type mice will be examined for their response to phlebotomy or phenylhydrazine-induced stress erythropoiesis. Stressed and unstressed mice will be analyzed for changes in erythroid parameters in peripheral blood (RBC counts, hematocrit, and hemoglobin), size of the spleen (the site of stress erythropoiesis in mice), and erythroid progenitor potential of cells isolated from adult bone marrow and spleen. In addition, the rate of cell division and expression of key cell cycle, erythroid regulators, and genes associated with stress erythropoiesis will be analyzed. Key findings from these studies will be validated using primary human CD34+ erythroid progenitors cultured under normoxic and hypoxic conditions in the presence of VDR shRNA lentiviruses or controls to knock down expression of VDR. Successful completion of the objectives outlined in this proposal could identify novel mechanisms and pathways that regulate erythropoiesis and lead to new approaches to treat anemia.